Use of ancient wheat crops for the diet

of non-celiac gluten sensitive patients

9th PROBIOTICS, PREBIOTICS & NEW FOODS, NUTRACEUTICALS AND BOTANICALS for Nutrition &

Human and Microbiota Health Roma, 10/12 Settembre 2017

Giuseppe Mazzarella

Institute of Food Sciences-CNR - Avellino

Biomarkers

YES

YES

NO

Gluten Related

Disorders

Pathogenesis

Autoimmune Allergic

Not Autoimmune

Not allergic

(Innate immunity?)

Celiac DiseaseGluten

Ataxia

Dermatitis

herpetiformis

Wheat

allergy

Gluten

sensitivity

Respiratory

Allergy

Food

AllegyWDEIA

Contact

UrticariaSymptomitic Silent Potential

Proposed New Classification of Gluten Related Disorders

Adaptive and + Innate Immunity

Frequency Intestinal Extra-intestinal

Very Common Bloating Lack of wellbeing

Abdominal pain Tiredness

Common Diarrhea Headache

Epigastric pain Anxiety

Nausea Foggy mind

Aerophagia Numbness

GER Joint/muscle pain

Aphtous stomatitis Skin rash/dermatitis

Alternating bowel habits Weight loss

Constipation Anemia

Loss of balance

Undetermined Hematochezia Depression

Anal fissures Rhinitis/asthma

Weight increase

Interstitial cystitis

Ingrown hairs

Oligo or polimenorrhea

Sensory symptoms

Disturbed sleep pattern

Hallucinations

Mood swings

Autism

Schizophrenia

The clinical manifestations of Non-Celiac Gluten Sensitivity (NCGS).

DIAGNOSIS

CD: serology ( anti-TG2) and histology ( villous atrophy)

WA: wheat specific IgE antibodies

NCGS: absence of specific diagnostic markers

To date, is mainly a “diagnosis of exclusion”

Pathogenesis of Celiac Disease (CD)

Maria I. Vazquez-Roque et al. Gastroenterology. 2013

Pathogenesis NCGS innate response?

Sapone A et al. Int Arch Allergy Immunol. 2010

Sapone A et al. BMC 2011

Duodenal IHC CD3+ IELs

40 X

A= Gluten Sensitive

B= Controls

C= CD Active

A B

C

Sapone A. et alt, Int. Arch. of Allergy and Immunology 2009

A small increased of

CD3+ intraepithelial

lymphocytes (IELs)

could be detected in

some NCGS patients,

compared to controls,

Research is intense to find

wheat varieties with absent or

low toxicity to be implemented

in new strategies for treatment

and prevention of CD. Among

candidates, there are ancient,

diploid wheat species for their

low capability to activate

intestinal T cell responses in

CD patients

The genealogy of cultivated members of the Triticum

family

Values reported in the literature within and among wheat types for celiac reactivity

Molberg and others 2005; Pizzuti and others 2006; Vincentini and others 2007, 2009; van den Broeck and others 2010a, 2010b

Absence of immunotoxic 33-mer peptide in Triticum Monococcum Einkorn

0

500

1000

1500

2000

2500

3000

3500

0 50 100 150 200 250

ug/ml PT

pg/

ml I

FN-g

PT-Sag TG

PT-Monlis TG

PT-ID-331 TG

Treatment with gliadin peptides from two monococcumaccessions induces γIFN production from all intestinal

gliadin-specific T-cell lines.

Dose-response curves

0

20

40

60

80

100

120

140

CD

25

+ ce

lls/m

m2

lam

ina

pro

pri

a

**

*

*

0

10

20

30

40

50

60

CD

3+

ce

lls/m

m e

pit

elu

im

***

T. aestivum wheat giadin

T. monococcummonlis gliadin

T. monococcumID-331 gliadin

T. aestivum wheat giadin

T. monococcummonlis gliadin

T. monococcumID-331 gliadin

**

Lamina propria CD25+ cells IEL CD3+ cells

IMMUNOHISTOCHEMISTRY

Challenge with gliadin peptides from two monococcum accessions induced increase of IEL and LP CD25 cells in treated CD biopsies.

P TG PT- monlis PT- ID331 Medium

Medium PT-ID331 PT-Monlis

Gliadins from monococcum wheat Monlis but not those from monococcum wheat ID331 induced IL-15 expression in intestinal mucosa

from treated CD patients.

Our data show that the monococcum lines Monlis and ID331 activate the CD T cell response and suggest that

these lines are toxic for celiac patients. However, ID331 is likely to be less effective in inducing CD because of its

inability to activate the innate immune pathways.

Gianfrani C et al. Am J Clin Nutr 2012;96:1339–45

SUMMARY

Gliadin proteins

Pepsin (2h)

Chymotrypsin (4h)

Pepsin (30 min)

Trypsin, Chymotrypsin, Elastase, Carboxypeptidase

(1h)

Brush Border Membrane enzymes

(BBM) (2h)

Deamindation by tTGase (2h)

T-cell and biopsy assay

RP-HPLC analysis

Collected fraction

Chymotrypsin (16h)

nLC-MS/MS

Pepsin (30 min)

nLC-MS/MS

Immunassay Proteomics

Gliadinidentification

Data base search

Data base search

Identification of resistant peptides

Immunogenity evaluation

PC GD-BBM

Trypsin, Chymotrypsin, Elastase,

Carboxypeptidase (1h)

Brush Border Membrane enzymes

(BBM) (2h)

GD-BBM

Pt#CD301012

0

200

400

600

800

1000

1200

Inte

rfero

n-g

(pg

/ml)

PC GD-BBM I331-PC I331 GI-BBM Monlis PC Monlis GI-BBM

Sag

0

20

40

60

80

100

120

140

Sag-PC Sag GD-BBM ID331-PC ID331 GD-BBM

CD

25

+ ce

lls\m

m²

lam

ina

pro

pri

a

*

*

*A

p<0,005

p<0,003

p<0,05

0

2

4

6

8

10

12

14

Sag-PC Sag GD-BBM ID331-PC ID331 GD-BBM

CD

3+C

D2

5+

acti

vate

d T

ce

lls

*

*

*

p<0,005

p<0,005

p<0,001

Marked reduction of specific T-cell response to monococcum gliadin after the GD-BBM digestion

Marked reduction of T cell activation response to monococcum gliadin after the GD-BBM digestion

Sagittario

ID331

Monlis

w a/b g

MS-based analysis showed that several

Triticum monococcum peptides, including

known T-cell epitopes, were degraded

during the gastrointestinal treatment,

whereas many of Triticum aestivum gliadin

survived the gastrointestinal digestion.

SUMMARY

Mazzarella G et al. Mol Nutr Food Res. 2015 Sep;59(9):1844-54

Therefore our data confirm that monocoque grain is not a safe grain for celiac disease, but could

be a cereal candidate to represent a major food alternative for patients with NCGS and / or for

the prevention of celiac disease in subjects at risk for familiarity .

Dose response of IL-8 release by monocyte-

derived DCs stimulated with purified ATI .

LPS served as positive control

Non-gluten α -Amylase/Trypsin Inhibitor (ATI) family

are strong activators of innate immunity.

Junker Y et al J Exp Med2012;209(13):2395-408

ATI bioactivity determination in ancient and modern cereals

Victor F. Zevallos et al. Gastroenterology 2017;152:1100–1113

Triticum Monococcum as a healthy food

The properties of MonLis einkorn compared with a variety of common wheat:

•higher protein content;

•higher content of ash and trace elements (iron, zinc, magnesium, phosphorus, potassium etc.);

•low presence of saturated fatty acids;

•lower starch content and better digestibility;

•higher presence of antioxidants and yellow pigmentation (carotenoids, including beta-carotene, the precursor to vitamin A);

•higher content of tocols (vitamin E). These facts are relevant to the functional activities of cells and are efficient antioxidants

Therefore, these data suggest their potentiality as functional foods

Carmen GianfraniGianfranco MamoneVera Rotondi AufieroISA-CNR, Avellino

Salvatore AuricchioRiccardo Troncone Dept Pediatrics, and ELFIDUniv Federico II, Naples

Acknowledgments

Nicola GiardulloGaetano IaquintoU.O. Gastroenterolgy, HospMoscati, Avellino

Alessio FasanoMassachusetts General Hospital Harvard Medical School

Thanks for your attention